There is great incentive for developing low cost technologies for sequencing genomes. While significant advances have been made in conventional sequencing methods, the reductions in cost and time required to make genome sequencing more feasable require the development of new methodologies. Indeed many new and promising methods are under development. However, these new approaches are generally limited by the inability of DNA polymerases to synthesize DNA with modified deoxynucleotide triphosphates (dNTPs). We propose to use our recently developed activity-based selection system to evolve DNA polymerases that efficiently recognize these modified dNTPs, and thus overcome one of the most significant barriers to the development of the novel sequencing methods. The selection system is based on the co-display on phage of DNA polymerase libraries and their DNA substrates, such that mutants that recognize the modified dNTPs covalently attach a biotin-dUTP, allowing for their selective isolation. In addition to the selections, various modern gene shuffling approaches are proposed to generate polymerase diversity. The Specific Aims of the proposal are: Specific Aim 1: Evolve DNA polymerases that efficiently synthesize DNA using fluorophore-labeled dNTPs to enable novel synthesis-by-sequencing methodologies. Specific Aim 2: Evolve polymerases that efficiently incorporate reversibly fluorophore tagged dNTPs with 3'-O-allyl protecting groups and then continue synthesis after double deprotection. Specific Aim 3: Collaborate to evaluate evolved polymerases in actual sequencing applications. The evolution of polymerases better suited for sequencing applications, and their evaluation in the actual sequencing methodologies, promises to dramatically facilitate the development of the methods and help make genome sequencing more commonplace. [unreadable] [unreadable] [unreadable]